J. Phys. Chem. A 2000, 104, 4333-4337
4333
Proton Polarizability of Hydrogen-Bonded Network and its Role in Proton Transfer in Bacteriorhodopsin Jianping Wang and Mostafa A. El-Sayed* Laser Dynamics Laboratory, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400 ReceiVed: December 22, 1999; In Final Form: February 24, 2000
Room-temperature time-resolved step-scan Fourier Transform Infrared (FTIR) spectroscopy has been used to study the photocycle of native bacteriorhodopsin (bR) suspension in both H2O and D2O. The kinetics of the retinal isomerization, and that of the protonation/deprotonation of the proton acceptor, Asp85, are compared in the µs to ms time domain. It is found that hydrogen/deuterium (H/D) isotope exchange does not significantly affect the kinetics of the retinal isomerization and relaxation processes. However, the protonation/deprotonation processes of Asp85 COO- become slower in D2O. We also studied the kinetics of the continuum absorbance change in the 1850-1800 cm-1 frequency region, which has previously been proposed to correspond to the absorption of the delocalized proton that is involved in the proton transport to the surface during the photocycle. An H/D isotope shift of the frequency range of this continuum absorbance has been confirmed by the observation that the band in the 1850-1800 cm-1 disappears in the photocycle of bR in D2O. These results could support the previous proposal that the intramolecular proton release pathway consists of an H-bonded network. Our results also suggest that the two independent processes, the transfer of a proton from the Schiff base to Asp85 and the release of a different proton to the extracellular surface, are closely coupled events.
Introduction Bacteriorhodopsin (bR), the only protein in the purple membrane (PM) of Halobacterium salinarium,1 has a retinylidene chromophore that binds to the -amino group Lys216 through a protonated Schiff base and has been known as a lightdriven proton pump. The electrochemical gradient generated across the membrane can be used by the cell for ATP synthesis.2 The primary structure of bR contains only one polypeptide chain of 248 amino acid residues in the form of R-helices.3 Upon illumination, bR undergoes a cyclic photoreaction with an alltrans to 13-cis retinal isomerization and the translocation of protons from the cytoplasmic to the extracellular side of the membrane.4 In the photocycle of bR, a series of photointermediates, J, K, L, M, N, and O, have been spectroscopically identified,4-6 with lifetimes ranging from hundreds of femtoseconds to tens of milliseconds. Two amino acid residues involved in the proton translocation have been identified by site-directed mutagenesis. Asp85 is the proton acceptor from the protonated Schiff base,7 and Asp 96 is the proton donor to the Schiff base.8,9 In the L to M transition, a proton is transferred from the Schiff base to Asp 85, which remains protonated for a few milliseconds. Proton release is detected by pH-sensitive dyes10,11 bound to the extracellular surface in the tens of microseconds after the initiation of the photocycle. Thus, the proton appearing at the surface is not the proton that is dissociated from the protonated Schiff base. Another proton donor group, XH, must be involved. Possible candidates, such as Glu204 and Arg 82, have been suggested as such a proton-release group, XH.12,13 Recently, by using time-resolved FTIR, Rammelsberg et al.14 have reported that during the L to M transition, Glu204 is found * To whom correspondence should be addressed. Tel: (404) 894-0292. Fax: (404) 894-0294. Email:
[email protected].
to be protonated and, therefore, less likely to be the protonrelease group XH. On the other hand, these authors have found that the IR continuum absorbance in the 1850 and 1800 cm-1 frequency region changes on the time scale of proton pumping, and therefore, they have suggested that the intramolecular proton transfer occur via an H-bonded network to the surface of the protein. In general, the observation of a continuum absorbance indicates the presence of a proton delocalized over an H-bonded chain with large proton polarizability.15 Proton polarizabilities are caused by fluctuation and shifts of the proton within the H-bonded chain. Particularly, large proton polarizabilities are observed due to the collective motion of the protons within H-bonded chains. Therefore, it is possible to follow the kinetics of the polarizability change of a delocalized proton. Proton polarizabilities are about 2 orders of magnitude greater than the polarizabilities due to distortion of electronic systems. For an H-bonded system, either homoconjugated (A-‚‚‚A) or heteroconjugated (A-‚‚‚H+‚‚‚B), the direct origin of the intense IR continuum absorbance is the delocalized proton within an H-bonded network. The homoconjugated system gives a symmetrical double minimum proton potential well or a symmetrical broad, flat potential well along the A‚‚‚H‚‚‚A coordinate. The continuum absorbance band usually appears in the frequency region between the major modes of H-O-H vibrations in H-bond system.15 In the present study, to understand the detailed proton release pathway inside the membrane during bR photocycle, we have carried out the time-resolved FTIR of bR in both H2O and D2O. The continuum absorption in the 1850-1800 cm-1 range in the µs-ms time domain has been examined. Our results confirm the presence of a bleached continuum band in the region 18501800 cm-1, with its kinetics similar to that of the protonation of Asp85 in H2O, that does not exists in D2O.
10.1021/jp994460u CCC: $19.00 © 2000 American Chemical Society Published on Web 03/31/2000
4334 J. Phys. Chem. A, Vol. 104, No. 18, 2000
Wang and El-Sayed
Materials and Methods bR containing purple membrane fragments were isolated from the Halobacterium salinarium strain ET1001 as described previously.1 The membrane suspension in H2O or D2O was prepared by washing the membrane fragments 3-4 times by centrifugation at neutral pH. After centrifugation, the pellet was collected and squeezed between two CaF2 plates. The optical density of such a bR pellet is ∼1.0 at 570 nm. Samples were kept at 25 °C during experiments. The step-scan time-resolved FTIR system16 was an IFS 66/S system (Bruker, Billerica, MA), equipped with a MCT detector (Kolmar Technologies, mode KMPV11, with appropriate preamplifier for both ac- and dc-coupled signal output). The accoupled signal was amplified additionally by a factor of 125, using a DC-300 MHz amplifier (Stanford Research Systems, model SR 445), before inputting it to a 200 MHz digitizer. The dc-coupled signal of the detector was first adjusted to remove the offset and then amplified by a factor of 2-5, using a lownoise amplifier (Stanford Research Systems, SR 560), to approach (1V peak to peak amplitude before introducing it to the digitizer. The dc-coupled signal was used for phase correction when the ac-coupled transient spectra were collected. The MCT detector-preamplifier system gives a linear relationship between the signal output and the IR beam intensity, which guarantees no significant baseline shift in the obtained accoupled transient spectra. The dc-coupled single-beam spectra were collected separately. The time-resolved FTIR spectrum for a certain time domain after photoexcitation was calculated based on the ac-coupled transient spectrum and dc-coupled single beam spectrum: ∆Abs ) log (ac/dc +1). To cover the time range from µs to ms, two different experiments have been carried out, with a time resolution of either 1 µs or 10 µs and a spectral resolution of 4 cm-1. The spectral window is confined between 750 and 2000 cm-1 by using an antireflecting coated germanium band-pass filter. The results were obtained by averaging 1014 coadditions during the step-scan experiment and 6-9 separate experiments. The bR samples were photoexcited by using the second harmonic output (532 nm) of a Nd:YAG laser (QuantumRay, DCR-3) at a repetition rate of 10 Hz and a laser energy of 3 mJ/pulse. All of the measurements were carried out at 25 °C.
Figure 1. Time-resolved FTIR difference spectra of bR in H2O and D2O at room temperature. The spectra are taken at the delay time of 8 µs (L/bR, spectrum a for H2O and spectrum b for D2O) and 100 µs (M/bR, spectrum c for H2O and spectrum d for D2O). The spectra are vertically shifted with real scale shown.
Result Transient Spectra of L/bR and M/bR in H2O and D2O. Figure 1 gives the transient FTIR spectra of bR in H2O and D2O, obtained at delay times of 8 µs (L/bR, spectra a and b) and 100 µs (M/bR, spectra c and d). In the time-resolved difference spectra, the positive bands belong to the photocycle intermediates and the negative bands belong to the ground state (bR568). bR, in either H2O or D2O, has a complete photocycle and functions as a proton or a deuteron pump. The top two are typical features of L/bR difference spectra, whereas the bottom two are typical features of M/bR difference spectra. The fingerprint region of retinal is also shown at 1528 cm-1 for the CdC stretch mode and 1256, 1200, and 1166 cm-1 for the C-C stretch modes. From the L to M transition, it is clear that protonated aspartate groups that give a negative band at 1740 cm-1, upon L formation in H2O, disappear when M is formed. The positive band at 1186 cm-1 is a distinct feature for the formation of L, due to the C-C stretching mode of the C15C14 bond, which is adjacent to the protonated Schiff base. The positive bands at 1763 cm-1 in H2O and 1750 cm-1 in D2O can be assigned to the CdO of Asp85, and the ∼13 cm-1 difference in frequency from H2O to D2O is due to the isotope
Figure 2. Kinetics of retinal CdC stretching vibrational mode at 1528 cm-1, as an indicator of the photocycle, in the case of H2O (a) and D2O (b). Two traces are overlapped for each case, by using time resolution of 1 and 10 µs. Note that there is a minus sign of the absorbance change, in the case of H2O.
effect (OdC-OH vs OdC-OD). The appearance of this band is characteristic of the formation of the M intermediate. Time Courses of CdC Vibration at 1528 cm-1 in H2O and D2O. Figure 2 shows the kinetics of the bleach due to the CdC retinal stretching vibrational mode at 1528 cm-1, as an indicator of the photocycle, in H2O (Figure 2a) and in D2O (Figure 2b). The decrease in its intensity as a function of time reflects the all-trans to 13-cis retinal isomerization. The photoisomerization process is known to take place in
